Fig. 1
From: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly
a Inset shows how the interaction of proteins A and B enables their SmBiT and LgBiT fusions (Promega, USA) to be brought into close proximity to each other, allowing complex formation to occur and reconstitution of the active NanoBit (Promega, USA) luciferase. Graph shows the reconstituted luminescence activity of the various combinations of potential interacting N- and C-terminal-fused SmBiT and LgBiT constructs linked either to full-length eIF4E or the eIF4G604–646 fragment, respectively, co-transfected into HEK293 cells. Individual N- and C-terminal LgBiT-linked eIF4E and eIF4G constructs co-transfected with SmBiT-HALO served as negative controls. b Western blot analysis of HEK293 cells, either co-transfected with SmBiT-eIF4E and eIF4G604–646-LgBiT or with each construct alone in combination with empty vector DNA, in whole cell lysate (Left, WCL) and with m7GTP pulldown analysis (right). Mock control consisted of co-transfection of empty SmBiT and LgBiT vectors. eIF4E and SmBIT-eIF4E (highlighted with black arrows) or eIF4G604–646-LgBiT were detected by using anti-eIF4E and anti-NanoLuc antibodies. c SmBiT-eIF4E and eIF4G604–646-LgBiT constructs were either co-transfected together or separately co-transfected with corresponding empty vector DNA. Mock control consisted of co-transfection with both empty SmBiT and LgBiT vectors into HEK293 cells. d SmBiT-eIF4E was co-transfected with either eIF4G604–646-LgBiT or eIF4G604–646(YLL)-LgBiT constructs. e SmBiT-eIF4E and eIF4G604–646-LgBiT constructs co-transfected into HEK293 cells with either empty vector (mock) or vectors containing 4EBP1 wild-type or 4EBP1 mutants (as indicated). f 4EGi1 and 4E1RCat were titrated onto HEK293 cell co-transfected with SmBIT-eIF4E or eIF4G604–646-LgBiT. Luciferase activity was measured after 4 h. Luminescence signals were normalised with those obtained from DMSO vehicle-treated cells. Compound titrations were also normalised with respect to the counter screen titrations of the compounds against full-length NanoLuc (Additional file 1: Figure S1). All values represent mean ± SD (n = 3). The molecular mass of the protein marker is indicated in Kd